Integrin receptor inhibitors

ABSTRACT

Provided are compounds of formula (I)  
                 
 
     wherein A, Q, W, X, Y, Z, R 1  to R 4 , m and n are as defined herein. Compounds of the invention bind to α 4  integrin receptors and thereby inhibit binding of ligands for α 4  integrins which is useful for prophylactic and/or therapeutic treatment of diseases and conditions associated with α 4  integrins or their ligands.

FIELD OF THE INVENTION

[0001] The present invention relates to novel compounds useful astherapeutic, prophylactic or diagnostic agents having binding affinityto integrin receptors, in particular to α₄ integrins.

BACKGROUND OF THE INVENTION

[0002] The integrins are α/β heterodimeric cell surface receptorsinvolved in numerous cellular processes from cell adhesion to generegulation (Hynes, Cell 1992, 69, 11-25; Hemler, Annu. Rev. Immunol.1990, 8, 365-368). Several integrins have been implicated in diseaseprocesses and have generated widespread interest as potential targetsfor drug discovery (Sharar et al, Springer Semin. Immunopathology 1995,16, 359-378). In the immune system, integrins are involved in leukocytetrafficking, adhesion and infiltration during inflammatory processes(Nakajima et al, J. Exp. Med. 1994, 179, 1145-1154). Differentialexpression of integrins regulates the adhesive properties of cells anddifferent integrins are involved in different inflammatory responses(Butcher et al, Science 1996, 272, 60-66). The α₄ integrins, α₄β₁(VLA-4) and α₄β₇ (LPAM), are expressed primarily on monocytes,lymphocytes, eosinophils, basophils, and macrophages but not onneutrophils (Elices et al, Cell 1990, 60, 577-584). The primary ligandsfor α₄ integrins are the endothelial surface proteins mucosal addressincell adhesion molecule (MAdCAM) and vascular cell adhesion molecule(VCAM) with lower affinity (Makarem et al, J. Biol. Chem. 1994, 269,4005-4011). The binding of the α₄β₁ or α₄β₇ to MAdCAM and/or VCAMexpressed on high endothelial venules (HEVs) at sites of inflammationresults in firm adhesion of the leukocyte to the endothelium followed byextravasation into the inflamed tissue (Chuluyan et al, Springer Semin.Immunopathology 1995, 16, 391-404). Monoclonal antibodies directedagainst α₄β₁, α₄β₇, MAdCAM or VCAM have been shown to be effectivemodulators in animal models of chronic inflammatory diseases such asasthma (Simmons et al, Blood 1992, 80, 388-395), rheumatoid arthritis(RA) (Juliano et al, Current Opinion Cell Biology 1993, 5, 812-818), andinflammatory bowel diseases (IBD) (Laberge et al, Am. J. Respir. CritCare Med. 1995, 151, 822-829 and Barbadillo et al, Springer Semin.Immunopathology 1995, 16). While antibodies have shown efficacy theymust be administered parenterally and are inherently cumbersome toproduce. Accordingly, it would be desirable to provide small moleculecompounds which inhibit the interaction between α₄ integrins and ligandsMAdCAM and/or VCAM which would be useful for treatment of chronicinflammatory diseases such as arthritis, asthma, multiple sclerosis,Chrohn's disease, ulcerative colitis, and hepatitis C.

SUMMARY OF THE INVENTION

[0003] According to an aspect of the present invention there is providedcompounds of formula (I)

[0004] wherein

[0005] A is a 5 or 6 member, saturated or unsaturated carbocycle orheterocycle optionally substituted by oxo and R₄;

[0006] Q is alkyl, alkenyl or alkynyl optionally substituted withhalogen, carboxyl, alkyl or aryl, and wherein one or more carbon atomsare optionally replaced with O, N, NR₆, S, SO, or SO₂;

[0007] X is —CR₅— or —N—;

[0008] Y is H, —CHR₃—, —CR₃═, or a bond;

[0009] Z is H, —CHR₃—, ═CR₃—, —NR₃—, ═N—, O, S, SO, SO₂ or a bond,provided that when one of Y and Z is H then the other is also H;

[0010] W is —C(O)NR₆—, —NR₆C(O)—, —C(S)NR₆—, —NR₆C(S)—, NR₆, O, S, SO₂,—CH₂—, —C—, —NR₆SO₂—, —SO₂NR₆—, —OC(O)NR₆—, —NR₆C(O)O—, —OC(S)NR₆—,—NR₆C(S)O—, —S—C(S)NR₆—, —C(O)—, —NR₆C(O)NR₆— or —NR₆C(S)NR₆—;

[0011] R₁ is hydrogen or is selected from the group consisting of alkyl,alkenyl and alkynyl, each of which is optionally substituted withhydroxyl, halogen, amino, nitro, carboxyl, a carbocycle, or aheterocycle; or R₁ is a carbocycle or heterocycle optionally substitutedwith hydroxyl, oxo, halogen, amino, or nitro;

[0012] R₂ is selected from the group consisting of alkyl, alkenyl andalkynyl, each of which is optionally substituted with halogen, hydroxyl,oxo alkoxy, amino, nitro, carboxyl, carboxamido, acyl, acyloxy,amidinyl, guanidinyl, thiol, alkylthio, or one or more carbocycle orheterocycle optionally substituted with halogen, hydroxyl, oxo, alkoxy,amino or carboxyl; or R₂ is a carbocycle or heterocycle optionallysubstituted with halogen, hydroxyl, oxo, alkoxy, amino, nitro, carboxyl,acyl, acyloxy, alkyl, alkenyl, alkynyl or a carbocycle or heterocycleoptionally substituted with halogen, hydroxyl, oxo, alkoxy, amino orcarboxyl;

[0013] R₃ and R₄ are independently selected from the group consisting ofH, hydroxyl, halogen, amino, nitro, carboxyl, alkyl, alkenyl, alkynyl, acarbocycle and a heterocycle, wherein said alkyl, alkenyl, alkynyl,carbocycle and heterocycle groups are optionally substituted with one ormore substituents selected from the group consisting of hydroxyl,halogen, amino, oxo and carboxyl, and optionally one or more carbonatoms of said alkyl, alkenyl and alkynyl group is replaced with N, NR₆,O, S, SO or SO₂;

[0014] R₅ is H or alkyl, alkenyl or alkynyl optionally having a carbonatom replaced with O, N or NR₆, and optionally substituted with COOR₁;or R₅ together with the carbon atom from which it depends forms a doublebond to an adjacent carbon or nitrogen atom of Q; or R₅ together with anon-adjacent carbon or nitrogen atom of Q form a carbocycle orheterocycle;

[0015] R₆ is hydrogen, alkyl, alkenyl or alkynyl;

[0016] m and n are independently 1, 2 or 3; and salts, solvates andhydrates thereof.

[0017] In another aspect of the invention, there is providedpharmaceutical compositions comprising a compound of the invention and apharmaceutically acceptable carrier, excipient or adjuvant.

[0018] In another aspect of the invention, there is provided a method ofinhibiting binding of an α₄ integrin to a protein ligand comprisingcontacting said α₄ integrin with a compound of the invention.

[0019] In another aspect of the invention, there is provided a method oftreating a disease or condition mediated by α₄ integrin receptors orligands of α₄ integrin receptors in a mammal, the method comprisingadministering to said mammal an effective amount of a compound of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Compounds are provided having binding affinity for α₄ integrins,having the general formula (I)

[0021] wherein A, Q, W, X, Y, Z, R₁ to R₄, m and n are as definedherein.

[0022] Ring A is a 5 or 6 member, saturated or unsaturated carbocycle orheterocycle optionally substituted by oxo (═O) and R₄. By “carbocycle”is meant herein to be a mono- bi- or tricyclic ring system containing a4-16 carbon atom scaffold that is saturated, partially unsaturated orfully unsaturated including aromatic. In the context of ring A, suitablecarbocycles include cycloalkyl, cycloalkenyl and aryl. In a preferredembodiment, ring A is a carbocycle selected from the group consisting ofcyclopentyl, cyclohexyl and benzene. In a most preferred embodiment ringA is benzene. In another embodiment, ring A is a heterocycle. By“heterocycle” is meant herein to be a mono-, bi- or tricyclic ringsystem comprising a combination of 4-16 carbon and hetero atoms (i.e. N,O, and S, as well as SO and SO₂) that is saturated, partiallyunsaturated or fully unsaturated including aromatic. In the context ofring A, preferred heterocycles are 5 and 6 member monocycles.Particularly preferred ring A heterocycles include pyridine, pyran,pyrimidine, pyrazine, pyridazine, pyrole, furan, thiophene, imidazole,pyrazole, thiazole and triazole. It is appreciated that ring Aencompasses heterocycles in which the heteroatoms may be shared with thecentral ring, if present, and/or may be adjacent to X.

[0023] Q is a divalent alkyl, alkenyl or alkynyl linking groupoptionally substituted with halogen, carboxyl, alkyl or aryl, andwherein one or more carbon atoms are optionally replaced with O, N, NR₆,S, SO, or SO₂. In a preferred embodiment, Q is alkyl having 1 to 3carbon atoms or methylene groups in length, and more preferably lengthof 2 methylene groups. In another preferred embodiment, the methylenegroup adjacent to the group X is replaced with a nitrogen atom or NR₆and more preferably with an oxygen atom. In a particularly preferredembodiment, Q is —O—CH₂— wherein the oxygen atom is adjacent to the Xgroup.

[0024] X is a bridging group —CR₅— or —N— from which the group Qdepends. In a preferred embodiment, X is —CR₅— wherein R₅ is H. Inanother embodiment R₅ is alkyl, alkenyl or alkynyl optionally having acarbon atom replaced with O, N or NR₆, and is optionally substitutedwith COOR₁. By “alkyl”, “alkenyl” and “alkynyl” is meant herein to bestraight or branched aliphatic groups having 1-10 carbon atoms,preferably 1-6 and more preferably 1-4. Preferred alkyl groups aremethyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl.Preferably, R₅ is an alkyl group having 1 to 3 carbon atoms or methylenegroups in length and is substituted with COOR₁. More preferably, R₅ is—(CH₂)₂—COOR₁. In another embodiment, R₅ together with the carbon atomfrom which it depends forms a double bond to an adjacent carbon ornitrogen atom of Q. In yet another embodiment R₅ together with anon-adjacent carbon or nitrogen atom from the group Q form a carbocycleor heterocycle. In this context, a preferred embodiment is when R₅ andthe carbon atom in Q which is beta to the group X form a 1,3-dioxolanering. In a particularly preferred embodiment, the dioxolane ring isspiro at X and the two oxygen atoms are alpha to the X group.

[0025] Y is H, —CHR₃—, —CR₃═, or a bond and Z is H, —CHR₃—, ═CR₃—,—NR₃—, ═N—, O, S, SO, SO₂ or a bond, provided that when one of Y and Zis H then the other is also H. In a preferred embodiment, one of Y and Zis a bond while the other is —CHR₃— thereby forming a six member ringfused to ring A and the benzene ring of formula (I) resulting in atricyclic ring system. In another preferred embodiment Y and Z are both—CHR₃— thereby forming a seven member ring. In a more preferredembodiment, Y and Z are both H wherein ring A and the benzene ring offormula (I) are linked via group X rather than forming a fused tricyclicring system. In a more preferred embodiment, Y and Z are both a bondthereby forming a five member ring fused to ring A and the benzene ring,and in a most preferred embodiment the five member ring together withring A and the benzene ring of formula (I) form a fluorenyl ring.

[0026] W is —C(O)NR₆—, —NR₆C(O)—, —C(S)NR₆—, —NR₆C(S)—, NR₆, O, S, SO₂,—CH₂—, —C—, —NR₆SO₂—, —SO₂NR₆—, —OC(O)NR₆—, —NR₆C(O)O—, —OC(S)NR₆—,—NR₆C(S)O—, —S—C(S)NR₆—, —C(O)—, —NR₆C(O)NR₆— or —NR₆C(S)NR₆—. In aparticular embodiment W is the amido group —C(O)NR₆— wherein thenitrogen atom is adjacent to the benzene ring of formula (I), oralternatively —NR₆C(O)— wherein the carbonyl is adjacent to the benzenering. In another particularly embodiment W is the sulfonamido group—NR₆SO₂— wherein the sulfur atom is adjacent to the benzene ring, oralternatively —SO₂NR₆— wherein the nitrogen atom is adjacent to thebenzene ring. In the context where W is amido or sulfonamido, R₆ ispreferably H or C₁₋₄ alkyl and more preferably H or methyl. In anotherparticular embodiment, W is O. In a particularly preferred embodiment Wis NR₆ wherein R₆ is H or C₁₋₄ alkyl and particularly H or methyl.

[0027] R₁ is hydrogen or is selected from the group consisting of alkyl,alkenyl and alkynyl, each of which is optionally substituted withhydroxyl, halogen, amino, nitro, carboxyl, a carbocycle, or aheterocycle. By “amino” is meant herein to be a primary, secondary ortertiary amine substituted with alkyl, alkenyl, alkynyl, aryl, oraralkyl each optionally substituted as provided herein. By “carboxyl” ismeant herein to be —COOH as well as carboxy ester groups thereof and inparticular alkyl esters thereof. Alternatively R₁ is a carbocycle orheterocycle optionally substituted with hydroxyl, oxo, halogen, amino,or nitro. In a preferred embodiment R₁ is H or a group which isliberated in vivo to yield a free carboxy group —C(O)O⁻.

[0028] R₂ is selected from the group consisting of alkyl, alkenyl andalkynyl, each of which is optionally substituted with halogen, hydroxyl,oxo (═O), alkoxy, amino, nitro, carboxyl, carboxamido, acyl, acyloxy,amidinyl (—C(NH)NH— or —NHC(NH)—), guanidinyl (—NHC(NH)NH—), thiol,alkylthio, or one or more carbocycle or heterocycle optionallysubstituted with halogen (F, Cl, Br or I), hydroxyl, oxo, alkoxy, aminoor carboxyl. By “alkoxy” is meant herein to include —O-alkyl, —O-alkenyland —O-alkynyl wherein alkyl, alkenyl and alkynyl are as previouslydefined. By “acyl” is meant herein to be a substituted carbonyl(—C(O)—). Suitable acyl groups include alkanoyl, aroyl and aralkanoyl.Suitable acyloxy groups include alkanoyloxy, aroyloxy and aralkanoyloxy.In a particularly preferred embodiment R₂ is a naturally occurring ornon-naturally occurring D or L-amino acid residue in which the alphanitrogen or alpha amino group is optionally acylated. In particularlypreferred embodiments, W is NR₆ and R₂ is N-acetyl-tyrosine, D-tyrosine,phenylalanine, benzoyl, isonipecotoyl, 4-methoxyphenylacetyl,1-fluorenyl-carbonyl, 1-naphthoyl, 2-naphthoyl, 3-hydroxy-phenylalanine,3-iodo-tyrosine, 3-fluoro-tyrosine, 3-chloro-tyrosone,4-(4-hydroxyphenyl)-benzoyl, 2,3,5,6-tetrafluoro-tyrosine,6-hydroxynaphthoyl, 2-phenyl-3-(4-hydroxyphenyl)propanoyl andN-acetyl-3-(4-hydroxyphenyl)proline. In a more preferred embodiment R₂is a tyrosine amino acid reside and more preferably a tyrosine residuewherein the alpha nitrogen is acylated with acetyl (i.e. N-acetyltyrosine).

[0029] In another embodiment R₂ is a carbocycle or heterocycleoptionally substituted with one or more halogen, hydroxyl, oxo, alkoxy,amino, nitro, carboxyl, acyl, acyloxy, alkyl, alkenyl, alkynyl or acarbocycle or heterocycle optionally substituted with halogen, hydroxyl,oxo, alkoxy, amino or carboxyl. In a particular embodiment R₂ is aheterocycle substituted with a phenyl group which in turn is optionallysubstituted with one or more halogen, hydroxyl, alkoxy or carboxyl. In aparticularly preferred embodiment, the heterocycle is a pyrrolidine ringsuch that when W is an amido group —C(O)NR₆— or —NR₆C(O)— R₂ and Wtogether form a proline amino acid residue. The proline residue ispreferably substituted at its beta carbon (i.e. 3-position of thepyrrolidine ring) with a phenyl group which is optionallypara-substituted (i.e at its 4-position) with hydroxyl thereby forming aconstrained tyrosine residue. The phenyl group is optionally substitutedat one or more of the 2, 3, 5 and 6 positions with a halogen, inparticular iodo (I), chloro (Cl) or fluoro (I). Further, the nitrogenatom of the proline residue is optionally acylated, in particular withan alkanoyl group such as acetyl.

[0030] R₃ and R₄ are independently selected from the group consisting ofH, hydroxyl, halogen, amino, nitro, carboxyl, alkyl, alkenyl, alkynyl, acarbocycle and a heterocycle, wherein said alkyl, alkenyl, alkynyl,carbocycle and heterocycle groups are optionally substituted with one ormore substituents selected from the group consisting of hydroxyl,halogen, amino, oxo and carboxyl, and optionally one or more carbonatoms of said alkyl, alkenyl and alkynyl group is replaced with N, NR₆,O, S, SO or SO₂. In a particular embodiment R₃ and R₄ are H.Alternatively, one or both of R₃ and R₄ are a carboxy group —COOR₁linked to the ring from which it depends via a linking group such as analkyl chain of 1-6 methylene groups in length and preferably 1-3methylene groups. The linking group may depend from the ring by afunctional group such as O (alkoxy), NR₆ (amino), an amido group or asulfonamido group. In a preferred embodiment, both ‘m’ and ‘n’ are theinteger 1.

[0031] R₆ is hydrogen, alkyl, alkenyl or alkynyl. In a preferredembodiment R₆ is C₁₋₄ alkyl and more preferably methyl. In another morepreferred embodiment R₆ is H.

[0032] In accordance with a preferred embodiment, compounds of theinvention have the general formula (II):

[0033] wherein

[0034] Q is alkyl, alkenyl or alkynyl optionally substituted withhalogen, carboxyl, alkyl or aryl, and wherein one or more carbon atomsare optionally replaced with O, N, NR₆, S, SO, or SO₂;

[0035] R₁ is hydrogen or is selected from the group consisting of alkyl,alkenyl and alkynyl, each of which is optionally substituted withhydroxyl, halogen, amino, nitro, carboxyl, a carbocycle, or aheterocycle; or R₁ is a carbocycle or heterocycle optionally substitutedwith hydroxyl, oxo, halogen, amino, or nitro;

[0036] R₃ and R₄ are independently selected from the group consisting ofH, hydroxyl, halogen, amino, nitro, carboxyl, alkyl, alkenyl, alkynyl, acarbocycle and a heterocycle, wherein said alkyl, alkenyl, alkynyl,carbocycle and heterocycle groups are optionally substituted with one ormore substituents selected from the group consisting of hydroxyl,halogen, amino, oxo and carboxyl, and optionally one or more carbonatoms of said alkyl, alkenyl and alkynyl group is replaced with N, NR₆,O, S, SO or SO₂;

[0037] R₆ is hydrogen, alkyl, alkenyl or alkynyl;

[0038] R₇ is hydrogen, hydroxyl, halogen, alkyl, alkoxy or halogensubstituted alkyl;

[0039] R₈ is H, alkyl, alkenyl or alkynyl;

[0040] R₉ is H or NR₁₁R₁₁, wherein R₁₁ and R₁₁, are independently H,acyl or and amino acid residue; or one of R₁₁ and R₁₁, together with R₈form a heterocycle;

[0041] R₁₀ is O or S;

[0042] m and n are independently 1, 2 or 3;

[0043] p is an integer from 1 to 5;

[0044] and salts, solvates and hydrates thereof.

[0045] In a preferred embodiment R₇ is hydroxyl at the para-position(i.e. 4-position) of the phenyl group. Further R₇ substituents asprovided when ‘p’ is the integer 2, 3, 4 or 5, include 3-iodo, 3-fluoro,3-chloro and 2,3,5,6-tetrafluoro.

[0046] In particular embodiments, R₆ and R₈ are H and R₁₀ is oxygen.Alternatively, R₆ is methyl.

[0047] In a preferred embodiment, R₉ is NR₁₁R₁₁ wherein R₁₁, is H andR₁₁, is H or acyl, in particular alkanoyl i.e. acetyl. In anotherpreferred embodiment, R₉ is NR₁₁R₁₁, wherein one of R₁₁ and R₁₁,together with R₈ form a 5-member heterocycle while the other of R₁₁ andR₁₁, is H or C₁₋₄ alkanoyl. Preferably R₁₁ and R₈ together form apyrrolidine ring and R₁₁, is H or alkanoyl i.e. acetyl.

[0048] Particular compounds of the invention include:

[0049] and salts, solvates and hydrates thereof.

[0050] It will be appreciated that compounds of the invention mayincorporate chiral centers and therefore exist as geometric andstereoisomers. All such isomers are contemplated and are within thescope of the invention whether in pure isomeric form or in mixtures ofsuch isomers as well as racemates. Stereoisomeric compounds may beseparated by established techniques in the art such as chromatography,i.e. chiral HPLC, or crystallization methods.

[0051] “Pharmaceutically acceptable” salts include both acid and baseaddition salts. Pharmaceutically acceptable acid addition salt refers tothose salts which retain the biological effectiveness and properties ofthe free bases and which are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, carbonic acid, phosphoric acid and the like,and organic acids may be selected from aliphatic, cycloaliphatic,aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes oforganic acids such as formic acid, acetic acid, propionic acid, glycolicacid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid,maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid,citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilicacid, benzoic acid, cinnamic acid, mandelic acid, embonic acid,phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicyclic acid and the like.

[0052] Pharmaceutically acceptable base addition salts include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium and magnesium salts. Salts derived from pharmaceuticallyacceptable organic nontoxic bases includes salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic non-toxic bases are isopropylamine, diethylamine, ethanolamine,trimethamine, dicyclohexylamine, choline, and caffeine.

[0053] Compounds of the invention may be prepared according toestablished organic synthesis techniques from starting materials andreagents that are commercially available. In general, the compounds maybe prepared starting from a commercially available central ring system.Depending on the particular compound to be prepared, the central ringsystem is manipulated to append first the carboxylate moiety —Q—C(O)O—R₁to X and then the R₂—W— moiety to the benzene ring, or alternativelyfirst appending the R₂—W— moiety and then the —Q—C(O)O—R₁ moiety.

[0054] In a particular embodiment, compounds of formula (Ia), wherein Xis C and Q incorporates an oxygen adjacent to X (i.e. an ether), areprepared according to the general scheme 1.

[0055] Referring to scheme 1, starting compound (i), wherein A, Y, Z, W,R₂-R₄, m and n are as previously defined, is reduced with a suitablereducing agent, such as sodium borohydride (NaBH₄) or equivalenthydride, to give alcohol (ii). Depending on the particular substituents,starting compound (i) may be commercially available or is otherwiseprepared according to established organic synthetic techniques fromcompounds that are commercially available. The final ether compound (Ia)of the invention is achieved by treating alcohol (ii) with an base suchas NaH followed by reacting with elecrophilic intermediate (iii),wherein L is a suitable leaving group such as a halogen i.e. Br, and Q′is the same as Q, i.e. a carboxyl substituted alkyl, alkenyl or alkynylchain, minus the first carbon atom that would be adjacent to the ringsystem. It will be appreciated that depending on the particularsubstituents present in the compound, suitable protection anddeprotection procedures will be required as is standard in the art.Numerous protecting groups are described in Greene and Wuts, ProtectiveGroups in Organic Chemistry, 2d edition, John Wiley and Sons, 1991, aswell as detailed protection and deprotection procedures. For example,suitable amino protecting groups include t-butyloxycarbonyl (Boc),fluorenyl-methyloxycarbonyl (Fmoc), 2-trimethylsilyl-ethyoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), allyloxycarbonyl(Alloc), and benzyloxycarbonyl (Cbz). Carboxyl groups can be protectedas fluorenylmethyl groups and hydroxyl groups may be protected withtrityl, monomethoxytrityl, dimethoxytrityl, and trimethoxytrityl groups.

[0056] Preparation of compounds of the invention wherein X is carbon andQ is an alkyl chain, may be accomplished according to scheme 2.

[0057] Referring to scheme 2, starting compound (i) is reacted with anon-nucleophilic base such as NaH, LDA or TBAF, to give carbanion (ii)which is subsequently reacted with intermediate (iii) to yield compoundof formula (Ib) of the invention. In a particular embodiment wherein Qis and optionally substituted C₂ alkyl linker to —COOR₁, intermediatecarbanion (ii) is reacted with an α, β unsaturated carboxylate (iv)wherein each R is independently hydrogen, halogen, alkyl, carboxyl oraryl, to give compound (Ib) of the invention. Preferably R is halogen orC₁₋₄ alkyl i.e. methyl.

[0058] In another embodiment, compounds of formula (I), wherein X is N,are prepared according to scheme 3.

[0059] Referring to scheme 3, starting compound (i) is reacted withintermediate (ii) wherein L is a suitable leaving group, i.e. Br, in thepresence of a non-nucleophilic base such as Cs₂CO₃ or KCO₃ to yield thefinal compound (Ic) of the invention. Alternatively, starting compound(i) can be reacted with an α, β unsaturated carboxylate (iii) as inscheme 2 to give compound (Ic) of the invention.

[0060] In another embodiment, compounds of formula (I) wherein W is—C(O)NH—, —SO₂NH— or —NH— are prepared according to scheme 4.

[0061] Referring to scheme 4, from the starting amine compound (i) maybe prepared amide (Id), sulfonamide (Ie), amine (If), carbamate (Ig) andurea (Ih) by reacting (i) respectively with intermediates (ii) anactivated ester of R₂—CO₂H, (iii) R₂—SO₂—L wherein L is a leaving groupsuch as a halogen, (iv) R₂—L in the presence of a non-nucleophilic baseor R₂—C(O)H in the presence of NaCNBH₃, (v) R₂—OC(O)—L, and (vi)isocyanate R₂—N═C═O. It will be appreciated that thioamides may beprepared in a similar manner as the amides by using R₂—C(S)H orR₂—C(S)O—; and thiocarbamates may be prepared using R₂—S—C(O)—L orR₂—OC(S)—L; and thioureas may be prepared using isothiocyanate R₂—N═C═S.In a particular

[0062] In another embodiment, compounds of formula (I) wherein W is —O—or —S— may be prepared according to scheme 5a and 5b.

[0063] Referring to schemes 5a and 5b, the starting hydroxyl or thiolcompound is reacted with a non-nucleophilic base such as NaH to remove aproton. The anion is then reacted with R₂—L wherein L is a suitableleaving group to yield the resulting ether (Ii) and thioether (Ij)compound of the invention.

[0064] In another embodiment compounds of formula (I), wherein W iscarbonyl —C(O)—, may be prepared according to scheme 6.

[0065] Referring to scheme 6, starting compound (i) is reacted withaldehyde intermediate (ii) in the presence of NaCNBH₃ to give compound(Ik) of the invention.

[0066] In another embodiment, compounds of formula (I), wherein W is—NR₆SO₂—, are prepared according to scheme 7.

[0067] Referring to scheme 7, starting compound (i) is reacted withsulfuric acid and then POCl₃ to give the sulfonyl chloride compound(iii) which is reacted with R₂—NH₂ to give compound (Il) of theinvention.

[0068] In another embodiment, compounds of formula (I), wherein W is C,may be prepared according to scheme 8.

[0069] Referring to scheme 8, starting compound (i) is reacted with Pd°catalyst followed by R₂—B(OH)₂ or R₂—C≡CH to give final compound (Im) or(In) respectively.

[0070] In another embodiment, compounds of formula (I), wherein W iscarbamate —NR₆C(O)O— or thiocarbamate —NR₆C(S)O—, may be preparedaccording to scheme 9.

[0071] Referring to scheme 9, starting alcohol compound is reacted withintermediate R₂—NC(O)—L or R₂—NC(S)—L, wherein L is a suitable leavinggroup such as Br, to give final compound of the invention (Io) or (Ip)respectively. In a particular embodiment, a morpholino carbamatecompound of the invention by reacting the starting alcohol withmorpholine-C(O)—Cl.

[0072] In another embodiment, compounds of formula (I), wherein W isthiocarbamate —NR₆C(O)S— or thiocarbamate —NR₆C(S)S—, may be preparedaccording to scheme 10.

[0073] Referring to scheme 10, starting thiol compound (i) is reactedwith intermediate R₂—NC(O)—L or R₂—NC(S)—L, wherein L is a suitableleaving group such as Br, to give final compound of the invention (Iq)or (Ir) respectively.

[0074] In an aspect of the invention, there is provided a method ofinhibiting binding of an α₄ integrin to a ligand, the method comprisingcontacting said α₄ integrin with a compound of formula (I). The methodmay be carried out as a solution based or cell based assay wherein thecompound of the invention is introduced to the integrin in the presenceof a putative or known ligand of the integrin. The compound may belabeled, for example isotopically radiolabeled, to facilitate detectionof ligand binding or lack thereof to the integrin. Thus compounds of theinvention are useful for diagnostic assays.

[0075] Compounds of the invention are useful to prevent the interactionof an epithelial cell bearing VCAM-1 and/or MAdCAM on the cell surfacewith a leukocyte cell bearing α₄β₁ and/or α₄β₇ on the surface bycontacting the epithelial cell or the leukocyte with an inhibitoryamount of the compound of the invention. The compounds are useful inassays to determine the inhibitory effect of a compound whichantagonizes the binding of α₄β₁ and/or α₄β₇ integrin to VCAM-1 ligandand/or MAdCAM ligand. The inhibitory compound may be a small molecule, aprotein or peptide or an antibody. In an in vitro assay, the ligand orthe integrin may be directly or indirectly bound to a surface, such asmicrotiter plate, using known methods described for example in WO9820110, WO 9413312, WO 9624673, WO 9806248, WO 9936393, and WO 9910312.The other member of the binding pair, e.g. the integrin or the ligand,respectively, (or a cell expressing the same on its surface) is thenadded to the surface bound member and the inhibitory effect of a testmolecule is determined. The inhibitory activity of the compounds of theinvention can also be determined with this type of assay.

[0076] The binding of the integrins to their respective ligands is knownto be involved in inflammatory conditions associated with leukocyteinfiltration of tissues lined with epithelial cells expressing VCAM-1 orMAdCAM. Such tissues include the gastrointestinal tract, skin, urinarytract, respiratory airways and joint synovial tissues. The compounds ofthe invention are useful in treating diseases in which such binding isimplicated as a cause of the disease or symptoms of the disease.Undesired disease symptoms may arise from cell adhesion and/or cellactivation which releases proinflammatory mediators, typically whenthere is an increase or upregulation in the expression of VCAM-1 and/orMAdCAM on the surface of endothelial cells. Various disease states whichcan be treated and for which the inflammatory symptoms can be reducedupon administration of the compounds of the invention include rheumatoidarthritis, asthma, psoriasis, multiple sclerosis, inflammatory boweldisease including ulcerative colitis, pouchitis and Crohn's disease,Celiac disease, nontropical Sprue, graft-versus-host disease,pancreatitis, insulin-dependent diabetes mellitus, mastitis,cholecystitis, pericholangitis, chronic sinusitis, chronic bronchitis,pneumonitis, collagen disease, eczema, and systemic lupus erythematosis.Compounds of the invention are useful in treating these diseases andconditions by inhibiting the integrin/ligand binding.

[0077] Compounds of the invention are therapeutically and/orprophylactically useful for treating diseases or conditions mediated byα₄ integrin receptors i.e. α₄β₁ and α₄β₇, and/or their ligands, inparticular VCAM-1 and MADCAM-1. Accordingly in an aspect of theinvention, there is provided a method of treating a disease or conditionmediated by the α₄ integrin receptors or ligands of α₄ integrin receptorligands in a mammal, for example a human, comprising administering tosaid mammal an effective amount of a compound of the invention. By“effective amount” is meant an amount of compound which uponadministration is capable of reducing the amount of ligand able to bindto α₄ integrins in vivo; or an amount of compound which uponadministration is capable of alleviating or reducing the severity ofsymptoms associated with the disease or condition mediated by α₄integrins or ligands thereof.

[0078] The actual amount of compound administered and the route ofadministration will depend upon the particular disease or condition aswell as other factors such as the size, age, sex and ethnic origin ofthe individual being treated and is determined by routine analysis. Inmethods of the invention, the compound may be administered orally(including buccal, sublingual, inhalation), nasally, rectally,vaginally, intravenously, intradermally, subcutaneously and topically.Compounds will be formulated into compositions suitable foradministration for example with suitable carriers, diluents, thickeners,adjuvants etc. as are routine in the formulation art. Accordingly,another aspect of the invention provides pharmaceutical compositionscomprising a compound of formula (I) and a pharmaceutically acceptablecarrier, excipient or adjuvant.

[0079] Compositions of the invention may also include additional activeingredients. Dosage forms include solutions, powders, tablets, capsules,gel capsules, suppositories, topical ointments and creams and aerosolsfor inhalation. Formulations for non-parenteral administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic carrier substances suitable for non-parenteraladministration which do not deleteriously react with compounds of theinvention can be used. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohol,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like. The formulations can be sterilizedand, if desired, mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings flavorings and/oraromatic substances and the like which do not deleteriously react withcompounds of the invention. Aqueous suspensions may contain substanceswhich increase the viscosity of the suspension including, for example,sodium carboxymethylcellulose, sorbitol and/or dextran. Optionally, thesuspension may also contain stabilizers.

[0080] In a preferred embodiment, compounds of the invention areadministered via oral delivery. Compositions for oral administrationinclude powders or granules, suspensions or solutions in water ornon-aqueous media, capsules, sachets, troches, tablets or SECs (softelastic capsules or caplets). Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids, carrier substances or binders may bedesirably added to such formulations. Such formulations may be used toeffect delivering the compounds to the alimentary canal for exposure tothe mucosa thereof. Accordingly, the formulation can consist of materialeffective in protecting the compound from pH extremes of the stomach, orin releasing the compound over time, to optimize the delivery thereof toa particular mucosal site. Enteric coatings for acid-resistant tablets,capsules and caplets are known in the art and typically include acetatephthalate, propylene glycol and sorbitan monoleate.

[0081] Various methods for producing formulations for alimentarydelivery are well known in the art. See, generally Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990. The formulations of the invention can be converted ina known manner into the customary formulations, such as tablets, coatedtablets, pills, granules, aerosols, syrups, emulsions, suspensions andsolutions, using inert, non-toxic, pharmaceutically suitable excipientsor solvents. The therapeutically active compound should in each case bepresent in a concentration of about 0.5% to about 99% by weight of thetotal mixture, that is to say in amounts which are sufficient to achievethe desired dosage range. The formulations are prepared, for example, byextending the active compounds with solvents and/or excipients, ifappropriate using emulsifying agents and/or dispersing agents, and, forexample, in the case where water is used as the diluent, organicsolvents can be used as auxiliary solvents if appropriate.

[0082] Compositions may also be formulated with binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrates (e.g., starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulfate). Tablets may be coated bymethods well known in the art. The preparations may also containflavoring, coloring and/or sweetening agents as appropriate.

[0083] Formulations of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing predetermined amounts of the activeingredients; as powders or granules; as solutions or suspensions in anaqueous liquid or a non-aqueous liquid; or as oil-in-water emulsions orwater-in-oil liquid emulsions. A tablet may be made by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine, the activeingredients in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, preservative,surface active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredients therein.

EXAMPLE 1

[0084] Synthesis of Compound 5

[0085] Commercially available 2-amino-9-fluorenone 1 (10 mmol) wasdissolved in 100 mL of methanol and 0.38 g (10 mmol) of sodiumborohydride was added. The mixture was stirred at 25° C. for 1 h andconcentrated to dryness under vacuum. The residue was suspended in 100mL of saturated aqueous NaHCO₃ ⁻ and extracted with ethylacetate (100mL). The ethylacetate layer was concentrated under vacuum and theresidue was crystallized from ethylacetate/hexane (2:1) to afford 1.4 gof pure 2-amino-9-hydroxyfluorene 2.

[0086] Compound 2 (0.39 g; 2 mmol) was dissolved in 50 mL of anhydrousDMF and cooled to 0° C. under nitrogen. Lithium bistrimethylsilylamide(2 mmol; 1M in THF) was added dropwise and stirring was continued for 30min. Iodoethylacetate (3 mmol) was added dropwise and the solution wasstirred 12 h at 0° C. The reaction mixture was poured into 200 mL ofwater and extracted with ethyl acetate (200 mL). The extract wasconcentrated and the product was purified via silica gel chromatography(25% ethylacetate/hexane) to afford 0.34 g of fluorenyl ether 3.

[0087] Fluorenyl ether 3 (0.28 g; 1 mmol) was dissolved in 5 mL of DMFand N-FMOC L-tyrosine (1 mmol) was added followed by HATU (1 mmol) andN-methylmorpholine (2 mmol). The reaction was stirred overnight at 25°C., poured into 50 mL water, and the product extracted withethylacetate. Concentration under vacuum afforded 0.5 g of crude 4 whichwas used directly in the next step without further purification.

[0088] Compound 4 (0.5 g) was dissolved in 1:1 THF/water and 3 mL LiOH(1M in water) was added. The reaction was stirred for 4 h at 25° C. andconcentrated under vacuum to remove most of the THF. The aqueoussolution was lyophilized and the crude product was purified by reverseHPLC (acetonitrile/water gradient) to afford 90 mg of pure 5 as the TFAsalt.

EXAMPLE 2

[0089] Synthesis of 2-amino Carbazole

[0090] Carbazole (100.24 g) was suspended in acetic anhydride (300 mL)with catalytic boron trifluoride etherate (0.65 mL) and the solutionrefluxed for 25 min. then cooled to 0° C. and the solid was thencollected and recrystallized from hexane to give 82.65 g9-acetyl-carbazole.

[0091] 9-Acetyl-carbazole (41.85 g, 0.2 mol) was dissolved in 1 Lmethylene chloride and 28.5 mL acetylchloride (0.4 mol) and 120 galuminum chloride (0.9 mol) were added and the solution refluxed for 1.5h. The solution was cooled to −78° C. and 6N HCl was added slowly whilestirring and allowed to warm to room temperature. Methylene chloride wasadded to dissolve the precipitate and the solution was extracted, andthe organics dried over Na₂SO₄, decolorized with charcoal, filtered andconcentrated. Recrystallization from benzene/hexane gave 27.9 g2,9-diacetylcarbazole.

[0092] 2,9-Diacetylcarbazole, (25.1 g, 0.1 mol) was dissolved in 100 mLpyridine. Hydroxylamine hydrochloride (10.42 g, 0.15 mol) was added andthe solution refluxed for 20 min. The mixture was cooled and poured into70 mL conc. HCl in ice. The precipitate was collected by filtration,washed with a large volume of water and dried in vacuum over KOH toyield 24 g white solid. 5 g of this solid (18.8 mmol) was dissolved in17 mL TFA and 0.3 mL trifluoroacetic anhydride (2.1 mmol) was added. Themixture was refluxed for 20 min then concentrated to give the crude2,9-diacetylcarbazole amine. Both acetyl groups were removed bysuspending the crude material in 400 mL 10% aq. KOH and refluxing for 1day. The solution was cooled and ethyl acetate added and the product2-aminocarbazole was extracted, washed with brine, dried over Na₂SO₄ andconcentrated.

EXAMPLE 3

[0093] Synthesis of Compound 6

[0094] To 0.1 g (0.55 mmol) 2-aminocarbazole in 6 mL DMF was added 0.37g (1.65 mmol) of N—Ac—L—Tyr, 0.37 g (1.65 mmol) HBTU and 0.3 mL (1.7mmol) DIPEA. The reaction was stirred for 1 h, poured into ethyl acetateand washed consecutively with 0.1N H₂SO₄, aq. NaHCO₃ and brine thendried over Na₂SO₄ and concentrated. Flash chromatography (10%methanol/methylene chloride) gave 0.16 g (0.26 mmol) of the acetylatedamine. This was stirred for 1 h with cesium carbonate (0.185 g, 0.57mmol) and ethyl bromoacetate (0.043 mL, 0.39 mmol) in 4 mL DMF and thenpoured into ethyl acetate and washed with saturated NH₄Cl and brine anddried over Na₂SO₄ and concentrated. Without purification this wasdissolved in 5 mL THF and 5 mL water and 0.043 g (1 mmol) LiOH wasadded. The ester was cleaved in <30 min and then diluted with ethylacetate and washed consecutively with 0.1N H₂SO₄, and brine and thendried over Na₂SO₄ and concentrated. The product was lyophilized andpurified by HPLC.

EXAMPLE 4

[0095] Synthesis of Compound 7

[0096] To 6.57 g (31.44 mmol) of the 9-acetylcarbazole in 200 mL CH₂Cl₂was added 10.68 g silver nitrate (63 mmol) and 25.1 g aluminum chloride(188.6 mmol). This was stirred at room temperature for 2 h, diluted withmethylene chloride and poured onto ice containing 82 mL conc. HCl. Theorganic layer was separated, washed three times with water, dried withMgSO₄ and concentrated to a dark brown solid. The solid was dissolved in200 mL methanol, 4% aq. KOH was added and the solution was refluxed for1 h. The solution was cooled and concentrated, then taken up in ethylacetate and washed consecutively with water and brine then dried overNa₂SO₄. The 9-nitrocarbazole (0.1 g, 0.47 mmol) was dissolved in 3 mLDMF and 0.13 mL (1.42 mmol) methyl acrylate and 0.69 g (2.12 mmol)Cs₂CO₃ were added. The reaction was stirred for 2 h, diluted with ethylacetate and washed with saturated NH₄Cl and brine and then dried overNa₂SO₄ and concentrated. Flash chromatography (25% ethyl acetate/hexane)gave 0.081 g alkylated 9-nitrocarbazole. This was dissolved in 5 mLmethanol and a spatula of Pd/C and a balloon of H₂ were used to reducethe nitrocarbazole to the amino carbazole. This was dissolved in 5 mLDMF and 0.167 g (0.75 mmol) N—Ac—L—Tyr, 0.394 g (0.96 mmol) HBTU and0.19 mL (1.12 mmol) DIPEA were added. This was stirred for 1 h, dilutedwith ethyl acetate and washed consecutively with 0.1N H₂SO₄, aq. NaHCO₃and brine and then dried over Na₂SO₄ and concentrated. Withoutpurification this was dissolved in 5 mL THF and 5 mL water and 0.063 g(1.49 mmol) LiOH was added. The ester was cleaved in <20 min and dilutedwith ethyl acetate and washed consecutively with 0.1N H₂SO₄, and brine.This was dried over Na₂SO₄ and concentrated and then lyophilized andpurified by HPLC.

EXAMPLE 5

[0097] Synthesis of Compound 8

[0098] To 0.3 g (1.64 mmol) 2-aminocarbazole in 6 mL DMF was added 1.2 g(6.59 mmol) of the constrained-N—Ac—L—Tyr, 2.31 g (6.1 mmol) HBTU and 2mL (11.5 mmol) DIPEA. The reaction was stirred for 2.5 h, poured intoethyl acetate and washed consecutively with 0.1N H₂SO₄, aq. NaHCO₃ andbrine and then dried over Na₂SO₄ and concentrated. Flash chromatography(10% methanol/methylene chloride) gave 0.60 g of the acetylated amine.0.20 g of this was stirred for 1 h with cesium carbonate (0.349 g, 1.07mmol) and ethyl bromoacetate (0.08 mL, 0.73 mmol) in 5 mL DMF which wasthen poured into ethyl acetate and washed with saturated NH₄Cl and brineand then dried over Na₂SO₄ and concentrated. Without purification thiswas dissolved in 4 mL THF and 4 mL water and 0.082 g (1.95 mmol) LiOHwas added. The ester was cleaved in <30 min and then diluted with ethylacetate and washed consecutively with 0.1N H₂SO₄, and brine. This wasthen dried over Na₂SO₄, concentrated and then lyophilized and purifiedby HPLC.

EXAMPLE 6

[0099] Synthesis of Compound 9

[0100] To 0.2 g (0.48 mmol) of the 2-aminocarbazole acylated by theconstrained-Tyr (as in example 5) in 5 mL DMF was added cesium carbonate(0.349 g, 1.07 mmol) and methylacrylate (0.043 mL, 0.73 mmol). This wasstirred for 1 h, then poured into ethyl acetate and washed withsaturated NH₄Cl and brine and then dried over Na₂SO₄ and concentrated.Without purification this was dissolved in 3 mL THF and 3 mL water and0.081 g (1.92 mmol) LiOH was added. The ester was cleaved in <20 min andthen diluted with ethyl acetate and washed consecutively with 0.1NH₂SO₄, and brine. This was dried over Na₂SO₄, concentrated and thenlyophilized and purified by HPLC.

EXAMPLE 7

[0101] Synthesis of 3-amino-dibenzosuberol

[0102] Dibenzosuberol (6.0 g, aldrich) was added to 100 ml of stirredconcentrated nitric acid at ambient temperature. Periodic reactionprogress analysis (tlc, NMR and IR) indicated that dibenzosuberol wasfirst oxidized to dibenzosuberone, then nitrated at the 3-position.After 2 hours, the reaction mixture was poured into ice water andextracted with 1:1 hexane:ethyl acetate. The crude product was purifiedby flash chromatography on silica (9:1 hex/EtOac) to yield 3.22 g (45%)of 3-nitrodibenzosuberone.

[0103] 3-nitrobenzosuberone (3.22 g) was reduced with hydrogen at 40 psiin 1:1 methanol/THF (40 ml), acetic acid (2 ml) and 10% Pd/C (0.5 g) for1.5 hours. Filtration and concentration gave 3-aminodibenzosuberone 1.35g (48%).

[0104] 3-aminodibenzosuberone (1.35 g) and sodium borohydride (3 g) weredissolved in 100 ml THF. Ethanol (30 ml) was added in portions over onehour with stirring at ambient temperature. After 18 hours, 1N HCl wasadded dropwise until a pH of 7 was obtained. The reaction mixture waspartitioned between 1:1 hexane/ethyl acetate and saturated aqueoussodium bicarbonate, washed with brine, dried over sodium sulfate,filtered and concentrated to give 1.0 g of crystalline3-aminodibenzosuberol (74%).

EXAMPLE 8

[0105] Synthesis of Compound 10

[0106] 3-aminodibenzosuberol (0.95 g) was dissolved in dry DMF (10 ml).Sodium hydride (0.25 g of 60% in oil) was added and the reaction mixturestirred until hydrogen evolution ceased. Ethyl iodoacetate (1.35 g) wasadded and the reaction mixture stirred at ambient temperature for 3hours then partitioned between ethyl acetate and water, washed withbrine, dried and concentrated. Flash chromatography on silica (75:25,hex:EtOAc) gave 0.79 g of 3-amino-5-(ethyl carboxymethyl)dibenzosuberane (60%).

[0107] 3-amino-5-(ethyl carboxymethyl) dibenzosuberane (395mg), N-acetyltyrosine (425 mg), HBTU (723 mg), diisopropylethylamine (491 mg) and 5ml anhydrous DMF were combined and stirred at ambient temperatureovernight. The reaction was partitioned between ethyl acetate and 10%aqueous citric acid. The organic phase was dried, filtered and theproduct purified on silica (96:4 methylene chloride:methanol) to give290 mg of 24015-94.

[0108] The above product (24015-94, 50 mg) was dissolved in 8 ml of 1:1ethanol:water and 6.1 mg of lithium hydroxide monohydrate added. After 3hours, hydrolysis was complete and 1 ml of acetic acid was added. Thereaction mixture concentrated and the product purified by HPLC (1 inchC-18, 10 to 90 in 60 min, 254 nm).

EXAMPLE 9

[0109] Synthesis of Compound 11

[0110] 68 μL of acrylic acid (0.25 mmol, R═H) was added to 4 mL of DCMfollowed by 135 mg of HOBt (0.25 mmol) and 157 μL of DIPC (0.25 mmol).After equilibration for 2 min this solution was combined with 200 mg ofWang polystyrene resin (0.5 mmol/g, 0.1 mmol) and allowed to shake for 4h at which time the resin was filtered and washed extensively withrepeating volumes of DMF, THF, MeOH, and DCM. In a dry vial, 2 mL of THFwas combined with 2 mL of a 1.0 M solution of TBAF in THF (AldrichChemical Company). 362.5 mg of 2-aminofluorene (X═H) was added to theTBAF solution and allowed to shake for 10 min. prior to adding it to thepreviously mentioned resin. The resin was stirred for 16 h and thewashed as before. At this point, the amino acid tyrosine was coupled tothe aniline on the fluorenyl ring by treating the resin with 4 mL of a0.25 M solution of the amino acid, HOAt, HATU, and DIPEA in DMF for 16h. After washing the resin the FMOC protecting group was removed bymixing the resin with 4 mL of a 25% piperidine solution in NMP for 10-15min. The piperidine was removed from the resin with repeated washingsand the acylation was performed on the resin using a solution of acetylanhydride and DIPEA each at a 0.25 M concentration in 4 mL of DCM for 1h, followed by a washing step. The compound was liberated from the resinby treating it with 4 mL of a 90% TFA, 5% DCM, 2.5% water and 2.5%triethylsilane cocktail for 1 h. The TFA solution was filtered from theresin and evaporated under a stream of nitrogen gas. The product waspurified on a C-18 reversed phase HPLC to yield 3 mg of product (R,R′═H) and confirmed by mass spectroscopy.

[0111] The reaction with the acrylate could also be performed where Rwas an alkyl group. In cases where R′═Br then Suzuki couplings usingaryl boronic acids were performed prior to the acylation withFMOC-tyrosine. A typical procedure is as follows. The resin (100 mg) isswelled in 2 mL NMP containing 0.25 M boronic acid, 0.25 M DIPEA, and 50mg of Palladium catalyst, Pd(PPh₃)₂Cl₂ and heated to 60° C. for 6 hours.A variety of amine or carbonate bases and paladium catalysts could bealso used to perform this transformation. Additionally the nitrogen onthe tyrosine ring could be acylated or sulfonylated under a variety ofconditions to yield products with functional groups other than theacetyl group. By products resulting from an additional acrylate addingto the 9-position of the fluorenyl ring were observed with higherloading resins and in some cases were isolated in significantquantities.

EXAMPLE 10

[0112] Biological Activity Assays

[0113] The compounds of the invention can be assayed for ability toblock the α₄β₇/MAdCAM-1 or α₄β₁/VCAM-1 binding interaction by additionof serial dilutions of the samples to plates with the receptors asfollows. 96-well plates are coated with mouse anti-human α₄ (31470D,PharMingen, San Diego, Calif.). The plates are decanted and blocked with0.5% BSA. After washing α₄β₁ or α₄β₇ is added, followed by incubationfor 2 h at room temperature. The plates are washed and samples of thesmall molecule antagonists are added to the plates with MAdCAM-1-Ig-HRPor VCAM-1-Ig-HRP for 2 h at room temperature. After an additional wash,the bound MAdCAM-1-Ig-HRP or VCAM-1-Ig-HRP is detected by addition oftetramethylbenzidine (TMB, Kirkegaard & Perry, Gaithersberg, Md.),followed by detection of the absorbance of the product.

[0114] Alternatively, the compounds can be assayed using any knownprotein-protein or cell-based assay method, such as those described, forexample, in WO 99/10312 (examples 179-180) and WO 99/36393 (RPMI-CS-1cell adhesion assay). See also Cardarelli et al., 1994, J. Biol. Chem.,269:18668-18673.

[0115] For example, 96-well ELISA plates are coated overnight at 4° C.with 2 μg/ml with anti-human CD49d, (31470D, PharMingen, San Diego,Calif.) in phosphate buffered saline. The plates are decanted andblocked with assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mMMnCl₂, 0.05% Tween-20 and 0.5% BSA) at room temperature for one hour,with gentle shaking. The plates are washed three times (in 50 mMTris-HCl, pH 7.5, 100 mM NaCl, 1 mM MnCl₂, 0.05% Tween-20) and 2 μg/mlof the desired integrin in assay buffer is added, followed by incubationat room temperature for two hours, with gentle shaking. After washingthree times, 50 μl of samples of the small molecule antagonists (serialdilutions from 10 mM stocks in 100% DMSO) are added to the plates with50 μl of 1 μg/ml MAdCAM-1-Ig-HRP or VCAM-1-Ig-HRP in assay buffer. Theplates are incubated two hours at room temperature, with gentle shaking,followed by washing six times. The bound MAdCAM-1-Ig-HRP orVCAM-1-Ig-HRP is detected by addition of the peroxidase substrate, 3,3′, 5, 5′, tetramethylbenzidine (TMB, Kirkegaard & Perry, Gaithersberg,Md.), for 10 minutes, followed by addition of 1M phosphoric acid to stopthe reaction. The absorbance of the solutions are read at 450 nm on aplate reader.

[0116] Suitable animal models exist for many diseases and conditionswhich can be treated with the compounds of the invention. Additionalconfirmation of the efficacy of these compounds in specific diseases andat desired doses can be assayed using these established models. Forexample, animal models of chronic inflammatory diseases such as asthma(Laberge, S. et al., Am. J. Respir. Crit. Care Med., 1995,151:822-829.), rheumatoid arthritis (RA; Barbadillo, C. et al., SpringerSemin. Immunopathol., 1995, 16:375-379), and inflammatory bowel diseases(IBD; Podalski, D. K., N. Eng. J. Med., 1991, 325:928-937; Powrie, F. etal., Ther. Immunol., 1995, 2:115-123) may be used to demonstrate theactivity of the compounds of the invention and to conduct dose andefficacy studies.

We claim:
 1. A compound of the formula (I):

wherein A is a 5 or 6 member, saturated or unsaturated carbocycle orheterocycle optionally substituted by oxo and R₄; Q is alkyl, alkenyl oralkynyl optionally substituted with halogen, carboxyl, alkyl or aryl,and wherein one or more carbon atoms are optionally replaced with O, N,NR₆, S, SO, or SO₂; X is —CR₅— or —N—; Y is H, —CHR₃—, —CR₃═, or a bond;Z is H, —CHR₃—, ═CR₃—, —NR₃—, ═N—, O, S, SO, SO₂ or a bond, providedthat when one of Y and Z is H then the other is also H; W is —C(O)NR₆—,—NR₆C(O)—, —C(S)NR₆—, —NR₆C(S)—, NR₆, O, S, SO₂, —CH₂—, —C—, —NR₆SO₂—,—SO₂NR₆—, —OC(O)NR₆—, —NR₆C(O)O—, —OC(S)NR₆—, —NR₆C(S)O—, —S—C(S)NR₆—,—C(O)—, —NR₆C(O)NR₆— or —NR₆C(S)NR₆—; R₁ is hydrogen or is selected fromthe group consisting of alkyl, alkenyl and alkynyl, each of which isoptionally substituted with hydroxyl, halogen, amino, nitro, carboxyl, acarbocycle, or a heterocycle; or R₁ is a carbocycle or heterocycleoptionally substituted with hydroxyl, oxo, halogen, amino, or nitro; R₂is selected from the group consisting of alkyl, alkenyl and alkynyl,each of which is optionally substituted with halogen, hydroxyl, oxo,alkoxy, amino, nitro, carboxyl, carboxamido, acyl, acyloxy, amidinyl,guanidinyl, thiol, alkylthio, or one or more carbocycle or heterocycleoptionally substituted with halogen, hydroxyl, oxo, alkoxy, amino orcarboxyl; or R₂ is a carbocycle or heterocycle optionally substitutedwith halogen, hydroxyl, oxo, alkoxy, amino, nitro, carboxyl, acyl,acyloxy, alkyl, alkenyl, alkynyl or a carbocycle or heterocycleoptionally substituted with halogen, hydroxyl, oxo, alkoxy, amino orcarboxyl; R₃ and R₄ are independently selected from the group consistingof H, hydroxyl, halogen, amino, nitro, carboxyl, alkyl, alkenyl,alkynyl, a carbocycle and a heterocycle, wherein said alkyl, alkenyl,alkynyl, carbocycle and heterocycle groups are optionally substitutedwith one or more substituents selected from the group consisting ofhydroxyl, halogen, amino, oxo and carboxyl, and optionally one or morecarbon atoms of said alkyl, alkenyl and alkynyl group is replaced withN, NR₆, O, S, SO or SO₂; R₅ is H or alkyl, alkenyl or alkynyl optionallyhaving a carbon atom replaced with O, N or NR₆, and optionallysubstituted with COOR₁; or R₅ together with the carbon atom from whichit depends forms a double bond to an adjacent carbon or nitrogen atom ofQ; or R₅ together with a non-adjacent carbon or nitrogen atom of Q forma carbocycle or heterocycle; R₆ is hydrogen, alkyl, alkenyl or alkynyl;m and n are independently 1, 2 or 3; and salts, solvates and hydratesthereof.
 2. A compound according to claim 1, wherein A is a benzenering.
 3. A compound according to claim 1, wherein Q is a alkyl chain of1 to 3 carbon atoms in length wherein the methylene group adjacent to Xis optionally replaced with oxygen.
 4. A compound according to claim 3,wherein said methylene chain is 2 carbon atoms in length.
 5. A compoundaccording to claim 4, wherein the methylene group adjacent to X isreplaced with oxygen.
 6. A compound according to claim 1, wherein R₁ isH.
 7. A compound according to claim 1, wherein X is —CR₅— and R₅ is H.8. A compound according to claim 1, wherein both Y and Z are hydrogen.9. A compound according to claim 1, wherein both Y and Z are a bondthereby forming a 5 member ring with X.
 10. A compound according toclaim 1, wherein W is —NR₆— and R₆ is H or C₁₋₄ alkyl.
 11. A compoundaccording to claim 10, wherein R₂ is a D or L-amino acid residue whereinthe alpha amino group is optionally an acyl group.
 12. A compoundaccording to claim 11, wherein said amino acid is tyrosine.
 13. Acompound according to claim 10, wherein R₂ is N-acetyl-tyrosine,D-tyrosine, phenylalanine, benzoyl, isonipecotoyl,4-methoxyphenylacetyl, 1-fluorenyl-carbonyl, 1-naphthoyl, 2-naphthoyl,3-hydroxy-phenylalanine, 3-iodo-tyrosine, 3-fluoro-tyrosine,3chloro-tyrosone, 4-(4-hydroxyphenyl)-benzoyl,2,3,5,6-tetrafluoro-tyrosine, 6-hydroxynaphthoyl,2-phenyl-3-(4-hydroxyphenyl)propanoyl andN-acetyl-3-(4-hydroxyphenyl)proline.
 14. A compound according to claim 1having the formula (II):

wherein Q is alkyl, alkenyl or alkynyl optionally substituted withhalogen, carboxyl, alkyl or aryl, and wherein one or more carbon atomsare optionally replaced with O, N, NR₆, S, SO, or SO₂; R₁ is hydrogen oris selected from the group consisting of alkyl, alkenyl and alkynyl,each of which is optionally substituted with hydroxyl, halogen, amino,nitro, carboxyl, a carbocycle, or a heterocycle; or R₁ is a carbocycleor heterocycle optionally substituted with hydroxyl, oxo, halogen,amino, or nitro; R₃ and R₄ are independently selected from the groupconsisting of H, hydroxyl, halogen, amino, nitro, carboxyl, alkyl,alkenyl, alkynyl, a carbocycle and a heterocycle, wherein said alkyl,alkenyl, alkynyl, carbocycle and heterocycle groups are optionallysubstituted with one or more substituents selected from the groupconsisting of hydroxyl, halogen, amino, oxo and carboxyl, and optionallyone or more carbon atoms of said alkyl, alkenyl and alkynyl group isreplaced with N, NR₆, O, S, SO or SO₂; R₆ is hydrogen, alkyl, alkenyl oralkynyl; R₇ is hydrogen, hydroxyl, halogen, alkyl, alkoxy or halogensubstituted alkyl; R₈ is H, alkyl, alkenyl or alkynyl; R₉ is H orNR₁₁R₁₁, wherein R₁₁ and R₁₁, are independently H, acyl or and aminoacid residue; or one of R₁₁ and R₁₁, together with R₈ form aheterocycle; R₁₀ is O or S; m and n are independently 1, 2 or 3; p is aninteger from 1 to 5; and salts, solvates and hydrates thereof.
 15. Acompound according to claim 14, wherein one of R₁₁ and R₁₁, togetherwith R₈ form a 5-member heterocycle while the other of R₁₁ and R₁₁, is Hor C₁₋₄ alkanoyl.
 16. A compound according to claim 15, wherein R₅ is OHat the para position.
 17. A compound according to claim 1 selected fromthe group consisting of

and salts, solvates and hydrates thereof.
 18. A method of treating adisease or condition mediated by α₄ integrin receptors or ligands of α₄integrin receptors in a mammal comprising administering to said mammalan effective amount of a compound of according to claim
 1. 19. A methodaccording to claim 18, wherein said disease or condition is selectedfrom the group consisting of rheumatoid arthritis, asthma, psoriasis,multiple sclerosis, inflammatory bowel disease including ulcerativecolitis, pouchitis and Crohn's disease, Celiac disease, nontropicalSprue, graft-versus-host disease, pancreatitis, insulin-dependentdiabetes mellitus, mastitis, cholecystitis, pericholangitis, chronicsinusitis, chronic bronchitis, pneumonitis, collagen disease, eczema,and systemic lupus erythematosis.
 20. A method of inhibiting binding ofan α₄ integrin to a protein ligand comprising contacting said α₄integrin with a compound of claim 1.